Calibrated analysis framework for nanopore direct RNA sequencing uncovers cell-specific m6A stoichiometry at conserved sites
Year: 2026
Type: preprint
Abstract: Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N6-methyladenosine (m6A) at the level of individual RNAs. However, a lack of systematic benchmarking continues to raise questions regarding the sensitivity, specificity, and reproducibility of this method. To address this and to establish a best-practice workflow, we evaluated multiple Dorado versions using in vitro transcribed RNA and an m6A methyltransferase inhibitor as specificity controls. We established that stringent filtering is necessary to reduce false-positive calls and found strong concordance at high-stoichiometry sites when compared to an orthogonal m6A mapping method (GLORI). Further, by applying DRS to human primary fibroblasts and HD10.6 neurons, we uncovered cell type-specific differences in m6A stoichiometry, indicating a finely tuned epitranscriptomic regulation. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m6A epitranscriptome to fibroblasts and neurons.
Source: eLife
Topic: RNA modifications and cancer
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W7160318669